ARTICLE IN PRESS

Journal of Biomechanics ] (]]]]) ]]]–]]]

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Review

Biomechanics of knee ligaments: injury, healing, and repair

Savio L.-Y. Woo, Steven D. Abramowitch, Robert Kilger, Rui Liang
Department of Bioengineering, Musculoskeletal Research Center, University of Pittsburgh, Pittsburgh, PA, 15219, USA
Accepted 20 October 2004

Abstract

Knee ligament injuries are common, particularly in sports and sports related activities. Rupture of these ligaments upsets the
balance between knee mobility and stability, resulting in abnormal knee kinematics and damage to other tissues in and around the
joint that lead to morbidity and pain. During the past three decades, significant advances have been made in characterizing the
biomechanical and biochemical properties of knee ligaments as an individual component as well as their contribution to joint
function. Further, significant knowledge on the healing process and replacement of ligaments after rupture have helped to evaluate
the effectiveness of various treatment procedures.
This review paper provides an overview of the current biological and biomechanical knowledge on normal knee ligaments, as well
as ligament healing and reconstruction following injury. Further, it deals with new and exciting functional tissue engineering
approaches (ex. growth factors, gene transfer and gene therapy, cell therapy, mechanical factors, and the use of scaffolding
materials) aimed at improving the healing of ligaments as well as the interface between a replacement graft and bone. In addition, it
explores the anatomical, biological and functional perspectives of current reconstruction procedures. Through the utilization of
robotics technology and computational modeling, there is a better understanding of the kinematics of the knee and the in situ forces
in knee ligaments and replacement grafts.
The research summarized here is multidisciplinary and cutting edge that will ultimately help improve the treatment of ligament
injuries. The material presented should serve as an inspiration to future investigators.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Biomechanics; Knee ligaments; Tissue engineering; Healing

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Anatomy, histological appearance and biochemical constituents of normal ligaments. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Tensile properties of ligaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1. Ligament anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2. Significant biological factors on the properties of ligaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Viscoelastic properties of ligaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4.1. The quasi-linear viscoelastic theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Continuum based viscoelastic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Corresponding author. Department of Bioengineering, Musculoskeletal Research Center, 405 Center for Bioengineering, 300 Technology Drive,
P.O. Box 71199, Pittsburgh, PA 15219, USA. Tel.: +1 412 648 2000; Fax: +1 412 688 2001.
E-mail addresses: ddecenzo@pitt.edu, slyw@pitt.edu (S.L.-Y. Woo).

0021-9290/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jbiomech.2004.10.025
 ARTICLE IN PRESS
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5. Healing of knee ligaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

5.1. MCL healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
5.2. Phases of ligament healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
5.3. New animal model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

6. New approaches to improve healing of ligaments—functional tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

6.1. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
6.2. Gene transfer and gene therapy . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
6.3. Cell therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
6.4. Biological scaffolds. . . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
6.5. Mechanical factors . . . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7. ACL reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7.1. Graft function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7.2. Graft incorporation and remodeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

8. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1. Introduction majority of ligament reconstructions yield good short-

term clinical results, 20–25% of patients experience
Injuries to knee ligaments are very common. It has complications including instability that could progres-
been estimated that the incidence could be at 2/1000 sively damage other knee structures (Aglietti et al., 1997;
people per year in the general population (Miyasaka et Bach et al., 1998; Daniel et al., 1994; Jomha et al., 1999;
al., 1991) and a much higher rate for those involved in Ritchie and Parker, 1996; Shelbourne et al., 1995; Yagi
sports activities (Bruesch and Holzach, 1993). Ninety et al., 2002).
percent of knee ligament injuries involve the anterior Thus, there has been a tremendous quest for knowl-
cruciate ligament (ACL) and the medial collateral edge to better understand ligament injuries, healing and
ligament (MCL) (Miyasaka et al., 1991). In fact, recent remodeling in hope to develop new and improved
studies have documented that ACL injuries in females treatment strategies. The needs in meeting this goal
are reaching epidemic proportions with the frequency of have stimulated researchers to seek new and innovative
rupture more than 3 times greater than that of their male methods of investigation. Because of the complex
counterparts (Anderson et al., 2001; Arendt and Dick, biological process, it has become clear that collabora-
1995; Powell and Barber-Foss, 2000). The results of tions from different disciplines rather than an indivi-
ligament injuries can be devastating. Frequently, surgery dualistic approach in research must be developed. In this
is required, but the outcomes are variable. Further, review, the properties of normal ligaments, including
post-surgical rehabilitation could require an extended their anatomical, biological, biochemical and mechan-
absence from work or athletic competition. ical properties, as well as the changes that occur
Basic science and clinical studies have revealed that a following injury will be described. The MCL will be
ruptured MCL can heal spontaneously (Frank et al., used as a model because of its uniform cross-sectional
1983; Indelicato, 1983; Jokl et al., 1984; Kannus, 1988). area, large aspect ratio, and propensity for healing.
However, laboratory studies have shown that its Subsequently, novel functional tissue engineering meth-
ultrastructure and biochemical composition remain odologies and some of the early findings will be
significantly altered (Frank et al., 1983; Niyibizi et al., presented. The challenging problems which remain to
2000; Weiss et al., 1991). Furthermore, the mechanical be solved and the potential of new treatment strategies
properties of the ligament substance remain substan- will be explored. In terms of ligament reconstruction, the
tially inferior to those of normal ligaments even after biomechanics of surgical reconstruction of the ACL and
years of remodeling (Loitz-Ramage et al., 1997; Ohland the utilization of robotics technology to study some of
et al., 1991). On the other hand, midsubstance tears of the key surgical parameters that affect the performance
the ACL and posterior cruciate ligament (PCL) would of the replacement grafts will be reviewed. It is hoped
not heal spontaneously and surgical reconstruction that these creative research approaches will inspire many
using a replacement graft is often required (Hirshman to join this course of investigation and ultimately help
et al., 1990; Kannus and Jarvinen, 1987). While the improve the treatment of ligament injuries.
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2. Anatomy, histological appearance and biochemical
constituents of normal ligaments
Ligaments are composed of closely packed collagen
fiber bundles oriented in a parallel fashion to provide for
stability of joints in the musculoskeletal system. The
major cell type is the fibroblast and they are interspersed
in the parallel bundles of collagen.
In the human knee, the MCL is approximately 80 mm
long and runs from the medial femoral epicondyle
distally and anteriorly to the posteromedial margin of
the metaphysis of the tibia. The lateral collateral
ligament (LCL) originates from the lateral femoral
epicondyle and passes postero-distally to the top of the
fibular head. The cruciate ligaments, which are named
anterior and posterior according to their site of
attachment to the tibia, are located within the capsule
and cross each other obliquely. The anterior cruciate
ligament (ACL) arises from the anterior part of the
intercondylar eminentia of the tibia and extends to
the posterolateral aspect of the intercondylar fossa of
the femur. The posterior cruciate ligament (PCL) arises
from the posterior part of the intercondylar eminentia of
the tibia and passes to the anterolateral aspect of the
intercondylar fossa of the femur. Although morpholo-
gically intraarticular, the cruciate ligaments are sur-
rounded by a synovial layer. The ACL consists of two
bundles, an anteromedial (AM) and a posterolateral
(PL) bundle. The AM bundle is thought to be important
as a restraint to anterior–posterior translation of the
knee, while the PL bundle is thought to be an important
restraint to rotational moments about the knee (Yagi
et al., 2002). This anatomic division of these bundles is
based on the gross tensioning pattern of the ACL during
passive flexion-extension of the knee, with the AM
bundle being tauter in flexion and the PL bundle tauter
in extension. The PCL is also composed of two distinct
bundles, the antero-lateral (AL) and the postero-medial
(PM) bundle. Additionally, ligaments are sometimes
found anterior and posterior to the PCL in some people.
They are the anterior meniscofemoral ligament (MFL;
i.e. ligamentum Humphrey) and the posterior menisco-
femoral ligament (i.e. ligamentum Wrisberg) (Girgis
et al., 1975).
Generally, ligaments are inserted to bone in two ways;
direct and indirect (Fig. 1). For direct insertions (e.g. the
femoral insertion of MCL), fibers attach directly into
the bone and the transition of ligament to bone occurs in
four zones: ligament, fibrocartilage, mineralized fibro-
cartilage and bone (Woo et al., 1987). For an indirect
insertion (e.g. the tibial insertion of MCL) superficial
fibers are attached to periosteum while the deeper fibers
are directly attached to the bone at acute angles (Woo
et al., 1987). The tibial insertion of the MCL crosses the
epiphyseal plate so that it can be lengthened in
synchrony with the bone growth.
3
bone
mineralized
fibrocartilage
fibrocartilage
ligament
(A)
deep fibers
bone
superficial
fibers
connects to
periosteum
(B)
Fig. 1. (A) Photomicrograph demonstrating direct insertion, i.e. the
femoral insertion of rabbit medial collateral ligament (MCL). (B)
Photomicrograph demonstrating indirect insertion, i.e. the tibial
insertion of rabbit MCL. (Hematoxylin and eosin, x50) (permission
requested from (Woo et al., 1987)).
Between 65 and 70% of a ligament’s total weight is
composed of water. On a fat-free basis, Type I collagen
is the major constituent (70–80% dry weight) and is
primarily responsible for a ligament’s tensile strength.
Type III collagen (8% dry weight) and Type V collagen
(12% dry weight) are other major components (Birk and
Mayne, 1997; Linsenmayer et al., 1993). Collagen Types
II, IX, X, XI, and XII have also been found to be
present (Fukuta et al., 1998; Niyibizi et al., 1996;
Sagarriga Visconti et al., 1996).
Variations in the concentrations of these basic
constituents lead to a diverse array of mechanical
behaviors of knee ligaments that are suitable for their
respective functions. A comparative study showed that
the tangent modulus and tensile strength of the rabbit
MCL is higher than the ACL (Woo et al., 1992) which
correlates with a larger mean fibril diameter for the
MCL (Hart et al., 1992). In addition, the fibroblasts of
the MCL are more spindle shaped (Lyon et al., 1991)
and produce a higher level of procollagen type I mRNA
(Wiig et al., 1991) and a lower collagen type III to type I
ratio in culture (Ross et al., 1990). Further, mechanical
loading has been found to regulate the gene expression
 ARTICLE IN PRESS
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of collagens in ligaments (Hsieh et al., 2002). Therefore,
each ligament’s composition is directly correlated with
its mechanical properties.
3. Tensile properties of ligaments
Ligaments are best suited to transfer load from bone
to bone along the longitudinal direction of the ligament.
Thus, their properties are commonly studied via a
uniaxial tensile test of a bone–ligament–bone complex
(e.g. femur-MCL-tibia complex). These tests result in a
load–elongation curve that is non-linear and concave
upward. This enables ligaments to help to maintain
smooth movement of joints under normal, physiologic
circumstances and to restrain excessive joint displace-
ments under high loads. The parameters describing the
structural properties of the bone–ligament–bone com-
plex include stiffness, ultimate load, ultimate elongation,
and energy absorbed at failure. With cross-sectional
area and strain measurements, a stress–strain curve
representing the mechanical properties (quality) of the
ligamentous tissue can be obtained. The parameters
describing the mechanical properties of the ligament
substance include tangent modulus, ultimate tensile
strength, ultimate strain, and strain energy density. A
large number of experimental methods have been
employed by investigators to overcome some of the
technical difficulties encountered in measuring the
mechanical properties of ligaments (Beynnon et al.,
1992; Ellis, 1969; Lam et al., 1992; Lee and Woo, 1988;
Peterson et al., 1987; Peterson and Woo, 1986; Smutz et
al., 1996). Furthermore, environmental factors can also
cause large differences in the experimental data obtained
(Crowninshield and Pope, 1976; Figgie et al., 1986;
a þ r1; (3)
Haut, 1983; Haut and Powlison, 1990; Noyes et al.,
1974). For more information on these methodologies
and environmental factors, the readers are encouraged
to read the provided references and study the chapter
entitled: Biology, Healing and Repair of Ligaments in
Biology and Biomechanics of the Traumatized Synovial
Joint: The Knee as a Model, 1992 by the authors (Woo
et al., 1992).
An equally important consideration is the geometry of
the ligament. Unlike the MCL whose cross-section is
relatively uniform over its length, the ACL and PCL
have two functionally distinct bundles that are loaded
non-uniformly (Fuss, 1989; Girgis et al., 1975; Sakane et
al., 1997). Thus, they need to be separated in order to
have a specimen with a more uniform cross-sectional
area for tensile testing. Using this approach, a study
performed at our center showed the tangent modulus of
a section of the rabbit ACL (516764 MPa) was less than
half of that for the rabbit MCL (11207153 MPa) (Woo
et al., 1992). Further, the tangent modulus, tensile
strength, and strain energy density of the AM bundle in
the human ACL was larger than that for the PL bundle
(Butler et al., 1992). In a separate study, the mechanical
properties of the bundles of the human PCL were found
to be different as well (Harner et al., 1995). The tangent
modulus of the AL bundle (2947115 MPa) was almost
twice that of the PM bundle (150769 MPa). The fact
that different bundles have different properties suggests
that each bundle contributes to knee joint stability
differently, which may have important ramifications on
their replacements (Table 1).
3.1. Ligament anisotropy
Ligaments are three dimensional (3-D) anisotropic
structures. To describe the 3-D mechanical behavior of
the human MCL, investigators have developed a quasi-
static hyperelastic strain energy model based on the
assumption of transverse isotropy (Quapp and Weiss,
1998). The total strain energy, W, in response to a
stretch along the collagen fiber direction, l; was defined
to be equal to the sum of the strain energy resulting from
ground substance (F1), collagen fibers (F2), and an
interaction component (F3),
W ðI 1 ; I 2 ; lÞ ¼ F 1 ðI 1 ; I 2 Þ þ F 2 ðlÞ þ F 3 ðI 1 ; I 2 ; lÞ (1)
where I1 and I2 are invariants of the right Cauchy stretch
tensor. For a uniaxial tensile test, F1 was described with
a two coefficient Mooney–Rivlin material model
F 1 ¼ 1=2½C 1 ðI 1  3Þ þ C 2 ðI 2  3Þ; (2)
where C1 and C2 are constants, and F2 was described by
separate exponential and linear functions. F3 was
assumed to be zero.
The Cauchy stress, T, can then be written as
T ¼ 2fðW 1 þ I 1 W 2 ÞB  W 2 B2 g þ lW l a
where, B is the left deformation tensor, and W1, W2, and
W l are the partial derivatives of strain energy with
respect to I1, I2, and l; respectively. The unit vector field,
a, represents the fiber direction in the deformed state,
and r is the hydrostatic pressure required to enforce
incompressibility.
It was found that this constitutive model can fit both
the data obtained from longitudinal and transverse
dumbbell shaped specimens cut from the human MCL
Table 1
Values for tangent modulus of the human MCL (Quapp and Weiss,
1998), AM and PL bundles of the human ACL (Butler et al., 1992),
and AL and PM bundles of the PCL (Harner et al., 1995).
Tangent modulus (MPa)
Human MCL Human ACL Human PCL
AM PL AL PM
332758 2837114 1547120 2947115 150769

S.L.-Y. Woo et al. / Journal of Biomechanics ] (]]]]) ]]]–]]]
Transverse
40 Longitudinal
30
Stress (MPa)
20
10
0
0 4 8 12
Strain (%)
Fig. 2. Stress–strain curves for human MCLs longitudinal and
transverse to the collagen fiber direction (permission requested from
Quapp and Weiss (1998)).
(Fig. 2). The longitudinal specimens displayed a tangent
modulus of 332.2758.3 MPa and a tensile strength of
38.674.8 MPa, while the transverse specimens were an
order of magnitude lower with a tangent modulus of
11.070.9 MPa and tensile strength of 1.770.5 MPa
(Quapp and Weiss, 1998).
3.2. Significant biological factors on the properties of
ligaments
The effects of immobilization and exercise on the
mechanical properties of ligaments has been investigated
by a number of laboratories (Larsen et al., 1987;
Newton et al., 1990; Noyes, 1977; Woo et al., 1987).
When rabbit hind limbs were subjected to a few weeks of
immobilization, there were marked decreases in the
structural properties of the femur–MCL–tibia complex
(FMTC). These decreases occurred due to subperiosteal
bone resorption within the insertion sites, as well as
microstructural changes in the ligament substance.
Remobilization was found to reverse these negative
changes. However, up to one year of remobilization was
required for the properties of the ligament to return to
normal levels following 9 weeks of immobilization (Woo
et al., 1987). Similar results were found for the
femur–ACL–tibia complex (FATC) of primates and
rabbits (Newton et al., 1990; Noyes, 1977). Long periods
of exercise training, on the other hand, only showed
marginal increases in the structural properties of
ligaments with a 14% increase in linear stiffness of the
FMTC and a 38% increase in ultimate load/body weight
(Laros et al., 1971; Woo et al., 1982, 1979). There was
only a slight change in the mechanical properties of the
ligament substance.
ARTICLE IN PRESS
5
Decrease Increase
Stress Stress
Immobilization Normal Exercise
Stiffness, and Strength
Activity
Tissue Mass,Tissue
16
In-vivo Loads and Activity Levels
Fig. 3. A schematic diagram describing the homeostatic responses of
ligaments and tendons in response to different levels of stress and
motion (permission requested from (Woo et al., 1987)).
Based on the results of these and other related studies,
a highly non-linear representation of the relationship
between different levels of stress and ligament properties
is depicted in Fig. 3. The normal range of physiological
activities is represented by the middle of the curve.
Immobilization results in a rapid reduction in tissue
properties and mass. In contrast, long term exercise
resulted in a slight increase in mechanical properties as
compared with those observed in normal physiological
activities.
Skeletal maturity also causes significant changes
to ligaments whereby the stiffness and ultimate load
of the FMTC was shown to increased dramatically
from 6 to 12 months of age followed by insignificant
change from 1 to 4 years in the rabbit model (Woo et al.,
1990). This corresponded with a change in failure
mode from the tibial insertion to the midsubstance
reflecting closure of the tibial epiphysis during matu-
ration (Woo et al., 1986). On the other hand, the
human FATC demonstrated a significant decrease in
the stiffness and ultimate load with increasing age
(Noyes and Grood, 1976; Woo et al., 1991). There-
fore, each ligament is unique in its growth, development,
and aging. Investigators should be cautious when
extrapolating age related changes from one ligament
(ex. ACL to PCL) or species (ex. rabbit to human) to
another.
4. Viscoelastic properties of ligaments
The complex interactions of collagen with elastin,
proteoglycans, ground substance, and water results in
the time- and history-dependent viscoelastic behaviors
of ligaments. In response to various tensile loading
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protocols, ligaments exhibit hysteresis (i.e. internal Recently, our research center has developed an alter-
energy dissipation), creep, and stress relaxation. The native approach whereby the QLV theory can be applied
following is a comprehensive review of the theories to to experiments which utilize a slow-strain rate in order
describe these properties. to avoid experimental errors such as overshoot and
vibrations (Abramowitch and Woo, 2004). Using
4.1. The quasi-linear viscoelastic theory Boltzmann’s superposition principle, it can be shown
that the loading portion of a stress relaxation experi-
The quasi-linear viscoelastic (QLV) theory developed ment with a linear strain history and strain rate, g; for
by Fung (Fung, 1993) is one of the most successful 0otot0 can be described by:
models to describe the time- and history-dependent ABg
viscoelastic properties of soft tissues (Carew et al., 1999; sðtÞ ¼
1 þ C lnðt2 =t1 Þ
Kim et al., 1999; Simon et al., 1984; Zheng and Mak, Z t
1999), especially ligaments (Abramowitch and Woo, f1 þ CðE 1 ½ðt  tÞ=t2 E 1 ½ðt  tÞ=t1 Þg
2004; Funk et al., 2000; Kwan et al., 1993; Woo et al., 0
1981) and tendons (Elliott et al., 2003; Thomopoulos eBgt @t: ð7Þ
et al., 2003). The theory assumes that a non-linear elastic
response and a separate time-dependent relaxation Similarly, the subsequent stress relaxation at a constant
function can be combined in a convolution integral to strain, from t0 to t ¼ 1; can be described by changing
result in a 1-D general viscoelastic model expressed as the upper limit of integration in Eq. (7) from t to t0,
follows: ABg
Z t sðtÞ ¼
qse ðÞ q 1 þ C lnðt2 =t1 Þ
sðtÞ ¼ Gðt  tÞ qt: (4) Z t0
1 q qt f1 þ CðE 1 ½ðt  tÞ=t2 E 1 ½ðt  tÞ=t1 Þg
The elastic response is a strain dependant function. 0
Bgt
One of the representations can be written as follows: e @t; ð8Þ

se ðÞ ¼ AðeB  1Þ: (5) where A; B; C; t1 ; and t2 are material constants to be

determined. Simultaneously curve-fitting these equa-
Using Fung’s generalized relaxation function based on
tions to the loading and relaxation portions of the data
the assumption of a continuous relaxation spectrum, the
from a stress relaxation experiment and assuming
time-dependent reduced relaxation function, G(t) (Fung,
ligaments are relatively insensitive to strain rate allows
1993), takes the form
the constants A, B, C, t1 ; and t2 to be determined
½1 þ CfE 1 ðt=t2 Þ  E 1 ðt=t1 Þg (Abramowitch and Woo, 2004). Because this approach
GðtÞ ¼ ; (6)
½1 þ C  Lnðt2 =t1 Þ accounts for relaxation manifested during loading, the
R1 errors in the obtained constants resulting from the
where E1 is the exponential integral, y ez =z dz; and,
assumption of an idealized step-elongation are mini-
C, t1 and t2 are constants with t1 5t2 :
mized.
Using this approach, the QLV theory has been
Recently, this approach was utilized to describe the
utilized to model the canine MCL (Woo et al., 1981).
viscoelastic behavior of the goat FMTC (Fig. 4). It was
Based on separate curve fitting of se ðÞ and G(t) to the
found that the obtained constants were improved
loading and relaxation portions of the experimental
compared to an approach that assumed an idealized
data, respectively, the constants of the QLV theory were
step-elongation. Specifically, constant t1 was found to
obtained. These constants were then employed to
be an order of magnitude lower using the new approach
successfully predict the peak and valley stress values of
which agrees with the results of a previous study that
a cyclic stress relaxation experiment of canine FMTCs.
analytically determined errors resulting from assuming
It should be noted, however, that the theory has been
an idealized step-elongation (Dortmans et al., 1984). In
developed based on the assumption of a idealized step-
addition, the obtained constants were verified by the
change in strain which is impossible to apply experi-
prediction of a second independent experiment whereby
mentally. Therefore, there are significant errors that
a more general cyclic strain history was utilized
could occur in determining the viscoelastic constants,
(Abramowitch and Woo, 2004).
especially t1 (Dortmans et al., 1984; Funk et al., 2000).
Previous methods to account for these errors include,
normalization procedures, iterative techniques, extra- 4.2. Continuum based viscoelastic models
polation and deconvolution, as well as directly fitting
the measured strain history (Carew et al., 1999; The QLV theory assumes that the rate of relaxation
Doehring et al., 2004; Funk et al., 2000; Kwan et al., remains relatively constant. Recent studies on ligaments
1993; Myers et al., 1991; Nigul and Nigul, 1987). from the rat and rabbit have shown that ligament
 ARTICLE IN PRESS
S.L.-Y. Woo et al. / Journal of Biomechanics ] (]]]]) ]]]–]]]
20
15
Stress (MPa)
10 Experimental Data
Theory
5
0
0 100 200 3500
Time (sec)
Fig. 4. A typical curve fit using the new approach to experimental data
obtained from a stress relaxation test of a goat FMTC (permission
requested from Abramowitch and Woo (2004)).
viscoelastic behavior is nonlinear (i.e. the rate of
relaxation decreases as the level of applied strain
increases up to 2.5% strain) (Hingorani et al., 2004;
Provenzano et al., 2001). In addition previous work has
demonstrated that the creep and stress relaxation
behaviors of the MCL likely arise from different
mechanisms (Thornton et al., 1997). In fact, Professor
Fung in his book Biomechanics (2nd ed; 1993) described
this phenomenon by suggesting ‘‘ycreep is fundamen-
tally more nonlinear, and perhaps does not obey the
quasi-linear hypothesis.’’ Thus, alternative viscoelastic
models, such as the single integral finite strain (SIFS)
theory, have been used to fully describe the 3-D
behavior of ligaments (Johnson et al., 1996). The theory
is based on the general integral series representation for
a nonlinear viscoelastic response (Pipkin and Rogers,
1968). The concepts of microstructural change resulting
from recruitment and fading memory to ensure that
more recent states of strain have greater weight in
determining the stress than earlier states are incorpo-
rated. The specific constitutive equation is written as:
T ¼  pI þ C 0 f½1 þ mIðtÞBðtÞ  mB2 ðtÞg
Z t
 ðC 0  C 1 Þ Gðt  sÞ
0 the MCL can be roughly divided into three overlapping
f½1 þ mIðsÞBðtÞ  mFðtÞCðsÞFT ðtÞg ds ð9Þ
where T is the Cauchy stress, p is the hydrostatic
pressure to enforce incompressibility, I is the identity
tensor, B is the left Cauchy–Green strain tensor, G(t) is
the time-dependant relaxation function, C0 is the
instantaneous modulus, and IðsÞ ¼ tr C, where C is the
right Cauchy–Green strain tensor. The SIFS model can
also be linearized to yield the equations for classical
linear viscoelasticity and reduces to an appropriate finite
elasticity model for time zero.
The model was applied to data from uniaxial
extension of younger and older human PTs and canine
7
MCLs (Johnson et al., 1996). Constants were deter-
mined from curve-fitting stress–strain and stress–relaxa-
tion data and used to predict the time-dependent stress
resulting from cyclic loading with good agreement.
Thus, SIFS theory can be used to model viscoelastic
behavior resulting from large deformations in 3-D. The
robustness of this theory makes it useful for many future
applications.
3600
5. Healing of knee ligaments
5.1. MCL healing
Because the injured MCL of the knee can heal
spontaneously, it has been used as an excellent experi-
ment model for many studies, especially those from the
rabbit (Weiss et al., 1991; Woo et al., 1987). These
studies have helped to understand that the rate, quality
and composition of the healing MCL are dependent on
the treatment modality. Conservative treatment of an
isolated MCL injury produced better results to those
with surgical repair either with or without immobiliza-
tion (Boorman et al., 1998; Weiss et al., 1991; Woo
et al., 1987). Immobilization after ligament injury was
shown to lead to a greater percentage of disorganized
collagen fibrils, decreased structural properties of the
FMTC, decreased mechanical properties of the ligament
substance, and slower recovery of the resorbed insertion
sites (Woo et al., 1987). Clinical studies have also
reported that patients with a complete tear of the MCL
respond well to conservative treatment without immo-
bilization by plastercasts (Fetto and Marshall, 1978). As
a result, the paradigm of clinical management has
shifted from surgical repair with immobilization to non-
operative management with early controlled motion
(Indelicato, 1995; Reider et al., 1994).
5.2. Phases of ligament healing
The continuous process of healing following a tear of
phases (Frank et al., 1983; Oakes, 1982; Weiss et al.,
1991). The inflammatory phase is marked by hematoma
formation which starts immediately after injury and
lasts for a few weeks. It is followed by the reparative
phase where fibroblasts proliferate and produce a matrix
of proteoglycan and collagen, especially type III
collagen, to bridge between the torn ends. Over the
next 6 weeks, increasingly organized matrix, predomi-
nantly type I collagen, and cellular proliferation occur.
Finally, the remodeling phase which is marked by
alignment of collagen fibers and increased collagen
matrix maturation can continue for years (Frank et al.,
1983).
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Thus, the constituents of the healing ligament are
abnormal even after one year (Weiss et al., 1991). It
contains increased amount of proteoglycans, a higher
ratio of type V to type I collagen, a decrease in the
number of mature collagen crosslinks, and fibrils with
homogenously small diameters (70 nm) (Niyibizi et al.,
2000; Plaas et al., 2000; Shrive et al., 1995). Frequently,
there is an increase in the number of collagen fibrils of
the healed ligament, but the diameters of these fibrils are
smaller than those of a normal ligament (Frank et al.,
1997).
These changes are reflected in the structural properties
of the healing FMTC which are inferior to controls at 12
weeks after injury (Weiss et al., 1991). However, by 52
weeks post-injury the stiffness of the injured FMTC
recovered, but the varus–valgus (V–V) rotation of the
knee remained elevated and the ultimate load of the
5.3. New animal model
Animals that are large in size and more robust in
activity level, such as the goat model, have also been
studied (Ng et al., 1995). The tensile properties of the
healing goat FMTC can achieve stiffness and ultimate
load that are closer to control values at earlier time
periods than the healing rabbit FMTC (Abramowitch
et al., 2003a). Yet, the tangent modulus and morphology
of the healing ligament for the goat and rabbit models
were not different, suggesting that both heal with a
similar quality of tissue.
In addition, viscoelastic experiments show that the
percentage of stress relaxation of the healing MCL
remained twice that of contralateral controls (Abramo-
witch et al., 2004). Using the QLV theory, it was found
that, the initial slope of the elastic response, constants
FMTC remained lower than those for the sham-
operated MCL (Inoue et al., 1990; Loitz-Ramage
et al., 1997; Ohland et al., 1991). Concomitantly, the
cross-sectional area of the healing ligament measured as
much as 212 times its normal size by 52 weeks (Ohno
et al., 1995). Thus, the recovery of the stiffness of the
FMTC is largely the result of an increase in tissue
quantity.
The mechanical properties of the healing MCL
midsubstance remain consistently inferior to those of
the normal ligament and do not change with time up to
one year (Ohno et al., 1995; Weiss et al., 1991) (Fig. 5).
In terms of the viscoelastic properties of the healing
MCL, there is increased viscous behavior, reflected by a
greater amount of stress relaxation or creep, for the first
3 months after injury. However, some studies suggested
that these values returned to normal levels after this time
period (Chimich et al., 1991; Woo et al., 1987), while
others suggested they remained increased (Newton et al.,
1990).
Sham
30
6 Weeks
12 Weeks
52 Weeks
Stress (MPa)
20
10
0
0 1 2 3 4 5
Strain (%)
Fig. 5. Stress–Strain curves representing the mechanical properties of
the medial collateral ligament substance for sham-operated and
healing MCLs at time periods of 6 (n ¼ 6), 12 (n ¼ 6), and 52
(n ¼ 4) weeks (permission requested from (Ohland et al., 1991)).
A  B; was nearly an order of magnitude lower for the
healing MCL. In addition, the healing MCL dissipated
more energy, had a longer recovery time upon removal
of load, and its long-term relaxation plateaued earlier as
dimensionless constant C was nearly 3 times greater for
healing MCLs and constant t2 was approximately 63%
of that for sham-operated controls.
Models to represent injuries to more than one
ligament, e.g. MCL & ACL, have also been studied.
Using the rabbit model, the healing MCL can benefit
from ACL reconstruction, but no long-term advantages
were found with primary repair of the MCL (Yamaji et
al., 1996). Thus, laboratory data have helped many
clinicians to choose to reconstruct the ACL and treat the
ruptured MCL non-operatively. Regardless, the struc-
tural properties of the FMTC, mechanical properties of
the healing MCL, and knee function all remained poorer
than those for isolated MCL injuries (Abramowitch et
al., 2003c). Clinical data also support these findings
(Yamaji et al., 1996).
6. New approaches to improve healing of ligaments—
functional tissue engineering
In order to improve the quality of healing tissues and
restore the normal function of ligaments, functional
tissue engineering based on novel biological and bioengi-
neering techniques has been explored. Examples include
the usage of a variety of growth factors, gene transfer and
gene therapy, cell therapy, as well as the use of
scaffolding materials. Together with mechanical factors,
these technologies offer great potential for the utilization
6 of functional tissue engineering in ligament healing.
6.1. Growth factors
By binding to their specific receptors on cell surfaces,
growth factors can arouse targeted biological responses.
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Studies have shown how the expressions of insulin-like
growth factor-I ( IGF-I ), transforming growth factor
(TGF-b), platelet-derived growth factor (PDGF), vas-
cular endothelial growth factor (VEGF) and fibroblast
growth factor (FGF) are altered in healing ligaments
and tendons (Duffy et al., 1995; Panossian et al., 1997;
Pierce et al., 1989; Schmidt et al., 1995; Sciore et al.,
1998; Steenfos, 1994).
In the early stages of MCL healing, three mammalian
isoforms of TGF-b1; b2 and b3; are involved in the
healing process. TGF-b1 is increased in and around the
wound site seven days following injury (Lee et al., 1998).
In vitro studies at our research center demonstrated that
the application of TGF-b1 increases collagen synthesis
1.5 fold over controls in both MCL and ACL fibroblasts
(Marui et al., 1997). TGF-b2 has been shown to increase
the expression of type I collagen at 6 weeks after injury,
resulting in a profound increase in healing mass, but
with limited increase in the structural properties of the
FMTC (i.e. the stiffness but not the load at failure of the
healing MCL could be increased) (Spindler et al., 2002,
2003).
PDGF could also play a significant role in the early
stages of healing as the application of PDGF-BB
improved the structural properties of the rabbit FMTC
between 2 and 6 weeks (Batten et al., 1996; Lee et al.,
1998). Similar results have been demonstrated in a rat
study (Batten et al., 1996). Locally applied PDGF may
also improve the mechanical properties of the ipsilateral
flexor tendon graft after ACL reconstruction (Weiler et
al., 2004).
The potential of synergistic effects of two or more
growth factors has been explored. A combination of
PDGF-BB/TGF-b1 did not enhance the structural
properties of the healing FMTC compared to the use
of PDGF-BB alone (Woo et al., 1998). In addition, the
PDGF/TGF-b2 combination also had no significant
effect compared to the use TGF-b2 alone (Spindler et
al., 2003). On the other hand, another study has shown
that combined local application of TGF-b1 and EGF
could improve the structural properties of the bone-
patellar tendon-bone autograft for ACL reconstruction
in canine (Yasuda et al., 2004). Clearly, the healing
process of ligaments is much more complex than the in
vitro cell culture environment and more studies are
necessary.
6.2. Gene transfer and gene therapy
Gene transfer using carriers including both retroviral
and adenoviral vectors as well as liposomes (Nakamura
et al., 1998) have been used to induce DNA fragments
into healing ligaments to promote or depress the
expression of certain genes in hope to improve their
quality.
9
In our studies, an adenoviral vector appeared to be
able to express more effectively in ligaments than
retroviral vectors. By using LacZ gene as a marker
gene, it was shown that the gene expression could last
for 6 weeks in ligaments with the use of adenovirus
(Hildebrand et al., 1999). In addition, an in situ gene
transfer of TGF-b1 using an adenoviral vector increased
the cellularity and enhanced the deposition of Type I
and III collagen in a ruptured ACL (Pascher et al.,
2004).
A promising method is antisense gene therapy using
oligonucleotides (ODNs) to reduce undesirable proteins
in the healing ligament. This methodology has been
shown to successfully reduce decorin in the healing
MCL of a rabbit resulting in increased diameters of the
collagen fibrils as well as an 85% increase in the tensile
strength of the healing MCL (Nakamura et al., 2000). In
our research center, antisense gene therapy was used to
reduce the higher level of collagen types III and V in the
healing MCL. Preliminary in vitro data revealed that the
gene expression of these collagens could be lowered by
approximately 40% (Jia et al., 2002, 2001; Shimomura
et al., 2002). In vivo studies showed that ODNs were
taken up by fibroblasts and reduced the expression of
the type V collagen protein. This is indeed a promising
and exciting approach that warrants additional studies.
6.3. Cell therapy
Cell therapy using mesenchymal progenitor cells
(MPCs) or mesenchymal stem cells (MSCs) also has
tremendous potential in tissue engineering. These cells
can differentiate into a variety of cell types, including
fibroblasts (Lazarus et al., 1995). MSCs isolated from
the bone marrow, cultured with or without gene
transfer, and finally transplanted to host tissues appear
to retain their potential to differentiate (Bruder et al.,
1997; Goshima et al., 1991; Haynesworth et al., 1992).
For the patellar tendon in rabbits, an autologous MSC-
collagen graft could improve the quality as well as
accelerate the rate of healing (Awad et al., 2003, 1999).
In our research center, it was found that MSCs
implanted in the injured MCL of the rat differentiated
into fibroblasts. In addition, the cells were found to have
migrated to the non-injured area of the ligament after 3
days. These results are encouraging because the MSCs
have the potential to serve as a vehicle for delivering
therapeutic molecules as well as directly enhance the
healing of ligaments (Watanabe et al., 2002).
6.4. Biological scaffolds
There are several biological scaffolds such as gels or
membranes made from alginate, chitosan, collagen or
hyaluronic acid (Drury and Mooney, 2003; Kim et al.,
1998). For ligaments, the porcine small intestinal
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submucosa (SIS) has been found to enhance their repair
(Badylak et al., 1999; Musahl et al., 2004). SIS is mainly
composed of collagen (90% of dry weight) and contains
a small amount of cytokines and growth factors such as
FGF and TGF-b (Badylak et al., 1999). It is a
resorbable scaffold that can hold cells and nutrients
necessary for healing as well as to provide a collagenous
structure to be remodeled (Badylak et al., 1995).
A study from our research center has demonstrated
the enhancement of the biomechanical properties and
biochemical compositions of healing ligament by using
SIS. The effect of a single layer of SIS treatment of a
6 mm gap injury of the rabbit MCL was examined at 12
and 26 weeks post-surgery. The stiffness of the FMTC
was found to increase 56% compared to the non-treated
control while the ultimate load also nearly doubled at 12
weeks post injury. Furthermore, the tangent modulus of
the healing MCL increased by more than 50% at 12
weeks and this effect persisted up to 26 weeks where the
SIS-treated group had a 33% higher tangent modulus
and a 49% higher stress at failure. The histological
appearance of the SIS treated MCL had increased
cellularity, greater collagen density, and improved
collagen fiber alignment (Musahl et al., 2004). Correla-
tively, the ratio of collagen type V/I was decreased with
a corresponding increase in collagen fibril diameter. All
the results indicate that the application of this potential
functional tissue engineering technology to enhance the
healing of ligaments is promising.
6.5. Mechanical factors
It is also well-known that mechanical environment
can induce changes in the cell behavior and collagen
architecture. In vitro, fibroblasts that were mechanically
Fig. 6. Randomly aligned cells cultured on a smooth dish (upper left). Aligned cells culture on dish etched with microgroove (upper right). Randomly
aligned matrix produced by cells cultured on a smooth dish (lower left). Aligned matrix produced by cells culture on dish etched with microgrooves
(lower right) (Wang et al., 2003).
stretched in a microgrooved substrate, i.e an environ-
ment designed to mimic the intact ligament, have the
tendency to align with the direction of stretch as well as
produce better organized collagen matrix (Fig. 6)
(Huang et al., 1993; Wang et al., 2003). Therefore,
functional tissue engineering with the application of
proper mechanical environment may lead to positive
changes in the mechanical properties of ligaments.
7. ACL reconstruction
It is hoped that the new knowledge gained from
studying and treating healing ligaments may one day
lead to alternative strategies for treating other ligaments
that do not heal (e.g. ACL and PCL of the knee). For
now, however, injuries to the ACL and PCL are
managed by ligament reconstruction using replacement
auto- or allografts. While many patients have benefited
from these transplantations, a large percentage
(20–25%) of patients for ACL reconstruction and a
higher percentage (up to 60%) for PCL reconstruction,
unfortunately, have less than satisfactory outcomes
(Lipscomb et al., 1993). Efforts are being made to better
understand the kinematics of the knee and the in situ
forces in the intact ACL and ACL replacement grafts.
To do this, the following section will review the
anatomical, biological and functional perspectives of
the intact ACL in comparison to current ACL
reconstruction procedures and grafts.
7.1. Graft function
Previous literature has documented many methods to
measure six degree of freedom (DOF) knee motion and
 ARTICLE IN PRESS
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the forces in ligaments and ligament grafts, i.e. buckle
transducers, implantable transducers, transducers at
ligament insertion sites, linkage systems, cutting studies,
etc. (Butler et al., 1980; Holden et al., 1994; Hollis et al.,
1991; Lewis et al., 1982; Markolf et al., 1990).
In general, translations are described as proximal–-
distal (d.PD), medial–lateral (d.ML), and anterior–poster-
ior (d.AP) translations, while rotations are referred to as
internal–external rotation (Y:IE), flexion–extension
(Y:FE), and varus–valgus (Y:VV) rotation. These mo-
tions are based on three anatomical axes: the axis of the
tibial shaft, the axis defined by the femoral insertion
sites of the collateral ligaments, and the floating axis
Fig. 7. Diagram detailing the joint motion description and the
translations and rotations for its three anatomical axes (adapted from
Woo et al., 1994, permission requested from Knee Surgery).
Fig. 8. Schematic drawing illustrating the six degrees of freedom of motion of the human knee joint.
11
perpendicular to these two axes (Fig. 7) (Chao, 1980;
Grood and Suntay, 1983).
It is very difficult to accurately control and reproduce
knee motion in all 6 DOFs. Therefore, previous studies
have been forced to constrain some of the degrees of
freedom of knee motion. Thus, data may not reflect the
true function of the knee ligaments. For example, it was
found that, when a valgus stress is applied to the
knee, the ACL, rather than the MCL, is the primary
restraint to varus–valgus rotation when the knee was
allowed five DOF of motion (angle of knee flexion was
fixed) (Markolf et al., 1976). However, if the ante-
rior–posterior translation and axial tibial rotations were
restricted (i.e. three DOF), then the role of the MCL,
and not the ACL was more dominant. It can be difficult
to compare results between different studies as the
degrees of freedom permitted during testing can have a
significant effect on the outcome (Ahmed et al., 1992,
1987; An et al., 1990; Barry and Ahmed, 1986; Lewis
et al., 1989, 1982).
About a decade ago, our research center developed a
robotic/universal force moment sensor (UFS) testing
system (Fig. 8) for the purpose of controlling and
reproducing the multiple degrees of freedom of knee
motion. This novel testing system has been used to
assess the function of the ACL and ACL grafts as well
as that of other ligaments and joints. To date, as many
as 65 studies have been published using this technology
(Woo et al., 1999) and many laboratories have recently
adopted this technology as well (Fujie et al., 2004; Gill
et al., 2003). The robotic/UFS testing system is capable
of applying external loads to knees, i.e. multiple and
combined loading conditions similar to those used
during clinical examinations (Daniel et al., 1985).
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Additionally, the robotic/UFS testing system can

quantitatively measure the in-situ forces in ligaments
and replacement grafts. The motions of the intact,
ligament deficient, and reconstructed knee can be
obtained with respect to the same reference position
(Ma et al., 2000). Most importantly, this advanced
methodology has the advantage of collecting experi-
mental data from the same cadaveric knee specimen
under different experimental conditions (such as ACL
intact, and ACL-reconstructed knee states), thus redu-
cing the effect of interspecimen variation and signifi-
cantly increasing the statistical power of the data
through the use of repeated-measures analysis of
variance for data analysis. In other words, even with a
large standard deviation, statistical significance can be
Fig. 9. Magnitude of the in situ forces in the intact anterior cruciate
demonstrated as long as the change in data is consistent ligament (ACL), anteromedial (AM) bundle and posterolateral (PL)
between each experimental condition. bundle under 134N of applied anterior tibial load (adapted from
The robotic/UFS testing system can operate in both Gabriel et al. (Gabriel et al., 2004)).
force and position control modes. While operating in
force control mode, the robot applies a predetermined
external load to the specimen and the corresponding Anterior Load Rotational Load
kinematics can be obtained. Alternatively, the robotic/
UFS testing system can operate under position control ACL Deficient
Knee
mode by moving the specimen along a previously *p<0.05
recorded motion path and the UFS records a new set 120 *
*
of force and moment data. The UFS is capable of
measuring three forces and three moments about and 100
Tibial Translation (%)

along a Cartesian coordinate system fixed with respect

Normalized Anterior

to the sensor. These forces and moments are then 80

translated to a point of application at the joint center in
order to determine the magnitude and direction of the
applied external loads (Fujie et al., 1995). Since the path
of motion can be precisely repeated with the robotic/
UFS testing system, the in situ force in a ligament can be
calculated by determining the changes in forces after
cutting a ligament, based on the principle of super-
position (Rudy et al., 1996).
Using this testing system, we have found that the
two anatomical bundles of the ACL (i.e. the anterome-
dial (AM) and posterolateral (PL) bundles) each
function individually even under the simplest loading
condition such as an anterior tibial load applied to the
knee (Fig. 9) (Sakane et al., 1997). We have also learned
that the ACL can resist anterior tibial translation in
response to a combined internal tibial torque and valgus
torque; therefore, in response to this combined rotatory
load, the knee undergoes anterior tibial subluxation
when the ligament is deficient (Fukuda et al., 2003;
Gabriel et al., 2004).
Currently, the majority of ACL reconstruction
procedures are performed by utilizing either the
ipsilateral bone-patellar tendon-bone or hamstring
tendon grafts. A study from our research center
comparing these two graft choices indicates that under
anterior tibial loads, both grafts were successful in
restraining anterior tibial translation when compared to
60
40
20
0
Hamstrings Patellar tendon
Fig. 10. Anterior tibial translation (mean7SD) in the reconstructed
knee (normalized to the deficient knee) in response to anterior tibial
load and combined rotational load at 301 of knee flexion (n ¼ 12)
(permission requested from (Woo et al., 2002)).
that of the ACL-deficient knee. However, under
rotatory loads, neither replacement graft was able to
reduce the anterior tibial translation significantly when
compared to that of the ACL-deficient knee (Fig. 10;
note that the black bars approach the dashed lined
which represents an ACL deficient knee). Although both
grafts were able to restore the in situ forces in the intact
ACL under anterior tibial loads, neither were successful
in restoring the in situ forces to those experienced by the
knee with an intact ACL under rotatory loads (Fig. 11;
in this figure the dashed line represents the in-situ force
in the intact ACL).
 ARTICLE IN PRESS
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Anterior Load Rotational Load tween different interfaces, i.e. bone to bone or tendon to
Intact ACL
bone interfaces (Grana et al., 1994; Jackson et al., 1993;
*p<0.05 Singhatat et al., 2002; Weiler et al., 2002; Weiler et al.,
* 2002). ACL reconstructions in a goat model using bone-
120 *
patellar tendon grafts offer the ability to study bone to
bone healing and soft-tissue to bone healing in the same
Normalized In Situ Force (%)

100
animal. Histological evaluations from 3 to 6 weeks
revealed progressive and complete incorporation of the
80
bone block in the femoral tunnel, but only partial
incorporation of the tendinous part of the graft in the
60
tibial tunnel.
In recent years, studies have aimed to enhance the rate
40
of integration of tendon-bone interfaces during early
graft incorporation that would permit an earlier and
20
more aggressive postoperative rehabilitation (Chen et
0
al., 2002). The use of bone morphogenic protein-2
Hamstrings Patellar tendon (BMP-2) has shown some potential (Martinek et al.,
2002) in both canine and rabbit models. The interface
Fig. 11. In situ force in the replacement grafts (normalized to the force
between the tendon graft treated with adenoviral-BMP-
in the intact ACL) in response to anterior tibial load and combined
rotational load at 151 of knee flexion (n ¼ 12) (permission requested 2-vector (AdBMP-2) and the bone was similar to the
from (Woo et al., 2002)). insertion of a normal ACL. Also, the stiffness and
ultimate load of the graft complexes were significantly
better for the AdBMP-2 treated grafts than for the
Based on the anatomy of the ACL, it appears that control grafts at eight weeks after surgery. Biological
common reconstructive procedures place the ACL grafts scaffolds, i.e. periosteum, have also been explored as an
too close to the central axis of the tibia and femur, thus interface between tendon and bone has shown some
making them inadequate for resisting rotatory loads success (Chen et al., 2002). All these results suggest an
(Kanamori et al., 2000; Woo et al., 2002; Yagi et al., exciting potential for clinical application. However,
2002). Therefore, more lateral graft placement that is there remains a need to identify the ideal growth factor
closer to the femoral insertion of the PL bundle has been and its dosage, as well as to consider any potential safety
examined (Kanamori et al., 2000; Woo et al., 2002). A concerns of using biological factors to augment bone-
series of studies from our research center were done to tendon healing.
find biomechanical solutions to this issue. First, it was Concerns of graft-tunnel motion have led to studies to
found that a more laterally placed graft yielded better evaluate the amount of motion that occurs in a
results, especially in resisting rotatory loads, even hamstring reconstruction using a titanium button and
though graft placement had little effect in resisting the polyester tape construct (Hoher et al., 1999). Shortening
anterior tibial load. the tape length from 35 to 15 mm could significantly
Second, an anatomic double bundle reconstruction reduce the motion by 33%, as 90% of this elongation
that replicates both the AM and PL bundle yielded resulted from the tape. A further study revealed that a
results that were closer to that of the intact knee when graft secured by a biodegradable interference screw can
compared to a single-bundle reconstruction (Yagi et al., shorten the effective length of the graft, thus minimizing
2002). These data have generated much clinical interest, the amount of graft-tunnel motion (Tsuda et al., 2002).
and surgeons, first in Asia and then in Europe, have In addition, it should also be noted that other factors
recently begun to adopt the anatomic double bundle including initial fixation strength (Kousa et al., 2003a,
reconstruction. Likewise, some surgeons in America b), tibial position during fixation (Hoher et al., 2001),
have recently begun to advocate this approach. and initial graft tension (Abramowitch et al., 2003b;
Yasuda et al., 1997) may influence graft tunnel motion,
7.2. Graft incorporation and remodeling the biological integration of the graft into the bone
tunnel, and ultimately ACL function.
Early graft incorporation and remodeling of ACL
grafts are essential to the success of ACL reconstruction.
This process is dependent on the cellular response to the 8. Future directions
mechanical forces applied to the graft during the healing
process and the amount of graft motion within the bone During the past three decades, significant advances
tunnel. Studies have demonstrated that the time for have been made in characterizing the biomechanical
complete graft incorporation differs significantly be- and biochemical properties of knee ligaments as an
 ARTICLE IN PRESS
14 S.L.-Y. Woo et al. / Journal of Biomechanics ] (]]]]) ]]]–]]]
individual component as well as determining the
contribution of ligaments to joint kinematics and
function. The tensile and viscoelastic properties of
ligaments, together with experimental and biologic
factors, have all helped to move the field forward.
Further, significant knowledge on the healing process
and replacement of ligaments after rupture can serve as
the basis for evaluating the effects of repair and
reconstruction.
This is indeed an exciting period for ligament
research. The new field of tissue engineering has offered
many possibilities (e.g. growth factors, gene transfer/
gene therapy, and biological scaffolds) to examine the
molecular and cellular response that can enhance the
healing tissue with improved properties. In our research
center, we believe a tissue engineered SIS scaffold can
further enhance the healing of ligaments. It is further
possible to improve this bioscaffold by seeding it with
ligament fibroblasts and then applying mechanical
conditioning to help the alignment of the collagen fibers
within the scaffold. Eventually, a combination of
seeding cells on a bioscaffold that is conditioned with
the ideal combination of mechanical stimuli and by the
roles of AS-ODNs for types V and III collagens could be
found to improve healing of ligaments. Indeed, there is
still a long way to go to translate cell responses to in
vivo situations and eventually to clinical application. As
the biology is so complex, it is evident that an approach
that involves the seamless integration of the fields of
biomechanics with other biological sciences is a neces-
sity. With that, improved outcomes in the process of
ligament healing may be expected. Furthermore, what is
Repeat on
Robotic/UFS
testing system
In situ
forces in
ligaments
Improvement
of Patient
Outcome
Fig. 12. Flow chart showing the utilization of in vivo kinematics data to drive experimental and computational methodologies leading to improved
patient outcome.
learned can be extended to other ligaments and tendons
that do not have the healing capability.
In terms of ligament reconstruction by replacement
grafts, it is time to move our focus towards in vivo
situations in order to optimize rehabilitation protocols
and provide athletes with an earlier return to sports.
While the robotic/UFS testing system has enabled us to
better understand the function of the knee ligaments and
has shown the road map to better ACL reconstruction,
important questions that remain include the identifica-
tion of the mechanism of ACL and other ligament
injuries, the best reconstruction procedures, and the
time course of healing and remodeling of the grafts.
Therefore, in vivo kinematics data will need to be
collected and then reproduced on cadaveric knees
utilizing the robotic/UFS testing system (Fig. 12). Major
efforts have been made in our research center on the
reproducibility of data when matching cadaveric knees
to groups of human subjects with similar knee laxity.
Thus, an estimate of the forces in the ACL during in
vivo activities may be obtained from cadaveric knees
using this novel methodology. Moreover, in vivo
kinematics can be integrated into computational mod-
els, and the in situ forces in ligaments during in vivo
activities can be determined. Once such a model is
validated through experimentation, it will be possible to
use the computational model to study complex external
loading conditions. These computational models can
also be used to develop a database containing the in situ
forces in ligaments, as well as the stress and strain data
for patients of different ages, genders, and sizes.
Furthermore, this technology and methodology can be
Kinematic
Data
In Vivo
Computational
model
∆ • In situ forces
in ligaments
• Stress/strain
data
Validation
Surgery Database
planning & (age, gender,
rehabilitation .
size, etc.)
 ARTICLE IN PRESS
S.L.-Y. Woo et al. / Journal of Biomechanics ] (]]]]) ]]]–]]] 15

FUTURE Abramowitch, S.D., Yagi, M., Tsuda, E., Woo, S.L.-Y., 2003c. The
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